Glioblastoma multiforme (GBM) is the most invasive and aggressive brain tumor in adults and children. Even with standard of care therapies such as chemotherapy, radiation and surgery 90% of GBM patients die within two years. Unfortunately, most targeted therapeutic strategies for GBM will fail to improve long-term patient survival due to tumor resistance arising from tumor heterogeneity and redundancy in oncogenic signaling pathways or to unanticipated toxicities in normal tissues. Among alternative strategies are ones that trigger pleotropic perturbations in key cancer molecular processes, more difficult for tumors to work around. Our group recently found one such pleotropic GBM-specific vulnerability in pre-mRNA 3' splice site (ss) recognition, from functional genetic screening for cancer-lethal genes in GBM stem-like cells (GSCs) and non- transformed neural stem cells (NSCs). Knockdown or partial chemical inhibition of 3' ss recognition triggered production of 100s of specific missplicing events (either intron retention or exon skipping) in essential genes only in GSCs, leading to cell cycle arrest and cell death. We have named these missplicing events CiSSRs for Cancer-induced 3' Splice Site Recognition defects. CiSSRs could be triggered in multiple human GBM isolates and subtypes, and when triggered in vivo profoundly regressed a human avatar GBM tumor, dramatically improving survival. The purpose of this grant is to reveal the underlying mechanism of GBM-specific CiSSRs and to develop actionable therapeutic strategies around them for GBM and other cancers. If successful, this grant will establish 3' ss recognition as a viable therapeutic target for GBM and likely other cancers, while delivering tangible therapeutic strategies. Aim 1 examines competing hypotheses regarding the underlying CiSSR biology, which will inform studies in tumors and clinical samples, including the possibility of finding prospective splicing tumor biomarkers. Aim 2 examines the novel finding that specific epigenetic regulators buffer loss of 3' ss recognition in GBM and speaks to recent reports of MYC synthetic lethality with BRD4 inhibition and also tests splicing inhibitor sudemycin D6 in GBM isolates. Aim 3 validates CiSSR biology and drug combinations in vivo and will identify new lead compounds that trigger CiSSRs in directly in adult and pediatric brain tumor isolates. In sum, this grant offers a GBM therapeutic program focused on splicing. The program interweaves mechanistic studies with pre-clinical trials and collaborations with leading researchers in splicing, splicing modulators, glioma tumor models, and small molecule discovery. One end- goal is to have sponsored clinical trials underway within the next 5-10 years.